Several contemporary methods exist for the analytical separation of components of a mixture. In general, a liquid sample containing compounds of interest is separated by partitioning between a mobile phase and a stationary phase, and the individual separated compounds are analyzed.
For example, electrophoresis, particularly capillary electrophoresis (“CE”) is a method for separation of individual molecular species from a mixture by the application of an electric field. Separation of the molecules occurs because of their different rates of movement through the solution, the rate being influenced by pH of the solution, the mass, and charge of the molecule, and the strength and duration of the electric field. For example, a separation is typically performed in a capillary tube that is filled with an electrically conductive electrolyte solution and that is open on both ends. An electric field is applied by high voltage electrodes arranged at the ends of the capillary. Detection of separated molecules may be performed in the capillary, e.g., by laser irradiation of fluorescent molecules through a window on the outer surface of a capillary produced by removing the polyimide coating. See, e.g., U.S. Pat. Nos. 5,312,535, 5,364,521, and 5,310,462.
High performance liquid chromatography (“HPLC”), is another method that employs partitioning between a mobile liquid phase under high pressure and a stationary phase, for example silica-based columns, including bonded silica, and organic resins such as divinyl benzene. Of these, reverse phase silica-based columns are preferred because they have high separation efficiencies, are mechanically stable, and a variety of functional groups can be easily attached for a variety of column selectivities. Recently, miniature HPLC chromatography systems and techniques have been developed. These techniques use columns of smaller internal diameter than are usually used in conventional HPLC separations, and they only require samples of less than about 1 μL. These techniques are referred to by several names, including “micro liquid chromatography” (or “MLC”), “micro-high-performance LC” or simply “micro LC,” “capillary LC,” or “nanoLC” (i.e., the term used herein). U.S. Pat. Nos. 4,102,782, and 4,346,610.
A newer method is capillary electrochromatography (“CEC”) in which an electric field is applied across capillary columns packed with microparticles and the resulting electroosmotic flow acts as a pump for chromatography. The technique combines the advantages of the high efficiency obtained with capillary electrophoretic separations and the general applicability of HPLC. CEC has the capability to drive the mobile phase through columns packed with chromatographic particles, especially small particles, when using electroosmotic flow. See, e.g., Colon, et al., J. Chromatog. 887, 43 (2000); Dadoo, et al., LC-GC 15, 630 (1997); Jorgenson, et al., J. Chromatog. 218, 209 (1981); Pretorius, et al., J. Chromatog. 99, 23 (1974); and U.S. Pat. Nos. 6,395,183, 5,378,334, 5,342,492, and 5,310,463. Generally, capillaries are packed, either electrokinetically or using a pump, with an appropriate stationary phase material, which may be a same stationary phase material commonly used in HPLC. The chromatography column generally comprises a fused silica capillary tube having a circular cross-section. A portion of the capillary column is packed with a stationary solid phase material (e.g., bonded silica particles about 1 to 3 μm in diameter) held in place with porous frits which are typically sintered silica particles disposed at the upstream (inlet) and downstream (outlet) ends of the column. Under the combined effects of mobile phase flow, ionic drift induced by the applied electric field, and the partitioning effect of the stationary phase, the mixture is separated. To avoid disturbing the stationary phase, detection and analysis of the various components of the mixture typically takes place in an unpacked, or open, portion of the capillary column adjacent the downstream end where the bands corresponding to the individual components of the mixture emerge from the packed capillary column.
The chromatography columns used in CEC, HPLC, and related analytical methods require for optimal performance a permeable containment devices to retain fluids or stationary phase material within a column, or to filter particles, e.g., particulate contaminants in analytical samples. Common containment devices include fiberglass packings, screens, and bonded particles, typically referred to as “frits.”
There are many different methods of making frits but most techniques employ the consolidation of small particles by sintering or melting compressed particles of a known size together. In one typical method, an appropriate material is ground up into small pieces and screened for a selected size range of particles. The particles are then compressed together in a mold and heated to fuse the particles together, but not to melt or degrade the particles. After heating, the material is further processed by machining, and welding or gluing to an appropriate substrate. Another approach uses filaments, of either metals and plastics, that are randomly arranged, compressed, and fused together. Such filamentous frits are generally only appropriate for large (i.e., non-capillary) columns. Yet another approach uses screens to provide a containment device that serves as an alternate to frits, but screens generally have a lower limit of performance based on the size of the wire or filament used. However, screens offer low back pressure compared to frits. Colon, et al., J. Chromatog. 887, 43 (2000).
Neither the frit nor the screen offers an ideal structure for the containment of a packing or for providing a particle filter in applications that require small hole or pore sizes, particularly for a packed capillary column as used in either liquid chromatography (“LC”) or capillary electrophoresis (“CE”). The conventional frit, because of the convoluted route of the pore including paths that contain lateral translations, has high back pressure. While a screen has low back pressure, the screen has a lower limit on pore size. Frits also cause a void volume that reduces the quality of chromatographic data, especially in smaller columns and in separations of small volumes in which the volume of the frit relative to the sample volume is considerable.
“Fritless” columns have been explored as an alternative to the shortcomings of frits and screens, particularly in CEC columns. For example, one report discloses in situ acrylate polymerization mediated by a free-radical mechanism. Chirica, et al., Anal. Chem. 72, 3605 (2000). Other reports demonstrate the in situ synthesis of frits by sintering of silicates. Chirica, et al., Electrophoresis 20, 50 (1999); Chirica, et al., Electrophoresis 21, 3093 (2000); see also Zeng, et al., Sensors and Actuators B 79, 107 (2001). Although these columns are mechanically stable, it is difficult to achieve a stable baseline. Others have attempted to make frits by in situ photopolymerization of acrylates. See Chen, et al., Anal. Chem. 72, 1224 (2000); Dulay, et al., Anal. Chem. 73, 3921 (2001); Chen, et al., Anal. Chem. 73, 1987 (2001); Kato, et al., J. Chromatog. A, 924, 187 (2001). The polymer is thought to act as a “nanoglue” by immobilizing the particles of the stationary phase material. Such photopolymerization methods are limited to the manufacture of columns which are optically transparent.